Calcium phosphate nanoclusters modify periodontium remodeling and minimize orthodontic relapse.

Orthodontic relapse is one of the most prevalent concerns of orthodontic therapy. Relapse results in patients' teeth reverting towards their pretreatment positions, which increases the susceptibility to functional problems, dental disease, and substantially increases the financial burden for retreatment. This phenomenon is thought to be induced by rapid remodeling of the periodontal ligament (PDL) in the early stages and poor bone quality in the later stages. Current therapies, including fixed or removable retainers and fiberotomies, have limitations with patient compliance and invasiveness. Approaches using biocompatible biomaterials, such as calcium phosphate polymer-induced liquid precursors (PILP), is an ideal translational approach for minimizing orthodontic relapse. Here, post-orthodontic relapse is reduced after a single injection of high concentration PILP (HC-PILP) nanoclusters by altering PDL remodeling in the early stage of relapse and improving trabecular bone quality in the later phase. HC-PILP nanoclusters are achieved by using high molecular weight poly aspartic acid (PASP, 14 kDa) and poly acrylic acid (PAA, 450 kDa), which resulted in a stable solution of high calcium and phosphate concentrations without premature precipitation. In vitro results show that HC-PILP nanoclusters prevented collagen type-I mineralization, which is essential for the tooth-periodontal ligament (PDL)-bone interphase. In vivo experiments show that the PILP nanoclusters minimize relapse and improve the trabecular bone quality in the late stages of relapse. Interestingly, PILP nanoclusters also altered the remodeling of the PDL collagen during the early stages of relapse. Further in vitro experiments showed that PILP nanoclusters alter the fibrillogenesis of collagen type-I by impacting the protein secondary structure. These findings propose a novel approach for treating orthodontic relapse and provide additional insight into the PILP nanocluster's structure and properties on collagenous structure repair.

Nanostructure-Mediated Transport of Therapeutics through Epithelial Barriers.

The ability to precisely treat human disease is facilitated by the sophisticated design of pharmacologic agents. Nanotechnology has emerged as a valuable approach to creating vehicles that can specifically target organ systems, effectively traverse epithelial barriers, and protect agents from premature degradation. In this review, we discuss the molecular basis for epithelial barrier function, focusing on tight junctions, and describe different pathways that drugs can use to cross barrier-forming tissue, including the paracellular route and transcytosis. Unique features of drug delivery applied to different organ systems are addressed: transdermal, ocular, pulmonary, and oral delivery. We also discuss how design elements of different nanoscale systems, such as composition and nanostructured architecture, can be used to specifically enhance transepithelial delivery. The ability to tailor nanoscale drug delivery vehicles to leverage epithelial barrier biology is an emerging theme in the pursuit of facilitating the efficacious delivery of pharmacologic agents.

Enhanced Prostate-specific Membrane Antigen Targeting by Precision Control of DNA Scaffolded Nanoparticle Ligand Presentation.

Targeted nanoparticles have been extensively explored for their ability to deliver their payload to a selective cell population while reducing off-target side effects. The design of actively targeted nanoparticles requires the grafting of a ligand that specifically binds to a highly expressed receptor on the surface of the targeted cell population. Optimizing the interactions between the targeting ligand and the receptor can maximize the cellular uptake of the nanoparticles and subsequently improve their activity. Here, we evaluated how the density and presentation of the targeting ligands dictate the cellular uptake of nanoparticles. To do so, we used a DNA-scaffolded PLGA nanoparticle system to achieve efficient and tunable ligand conjugation. A prostate-specific membrane antigen (PSMA) expressing a prostate cancer cell line was used as a model. The density and presentation of PSMA targeting ligand ACUPA were precisely tuned on the DNA-scaffolded nanoparticle surface, and their impact on cellular uptake was evaluated. It was found that matching the ligand density with the cell receptor density achieved the maximum cellular uptake and specificity. Furthermore, DNA hybridization-mediated targeting chain rigidity of the DNA-scaffolded nanoparticle offered ∼3 times higher cellular uptake compared to the ACUPA-terminated PLGA nanoparticle. Our findings also indicated a ∼ 3.7-fold reduction in the cellular uptake for the DNA hybridization of the non-targeting chain. We showed that nanoparticle uptake is energy-dependent and follows a clathrin-mediated pathway. Finally, we validated the preferential tumor targeting of the nanoparticles in a bilateral tumor xenograft model. Our results provide a rational guideline for designing actively targeted nanoparticles and highlight the application of DNA-scaffolded nanoparticles as an efficient active targeting platform.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Tissue factor targeting peptide enhances nanoparticle binding and delivery of a synthetic specialized pro-resolving lipid mediator to injured arteries.

Background: Specialized pro-resolving lipid mediators (SPM) such as resolvin D1 (RvD1) attenuate inflammation and exhibit vasculo-protective properties. Methods: We investigated poly-lactic-co-glycolic acid (PLGA)-based nanoparticles (NP), containing a peptide targeted to tissue factor (TF) for delivery of 17R-RvD1 and a synthetic analog 17-R/S-benzo-RvD1 (benzo-RvD1) using in vitro and in vivo models of acute vascular injury. NPs were characterized in vitro by size, drug loading, drug release, TF binding, and vascular smooth muscle cell migration assays. NPs were also characterized in a rat model of carotid angioplasty. Results: PLGA NPs based on a 75/25 lactic to glycolic acid ratio demonstrated optimal loading (507.3 pg 17R-RvD1/mg NP; P = ns) and release of RvD1 (153.1 pg 17R-RvD1/mg NP; P < .05). NPs incorporating the targeting peptide adhered to immobilized TF with greater avidity than NPs with scrambled peptide (50 nM: 41.6 ± 0.52 vs 32.66 ± 0.34; 100 nM: 35.67 ± 0.95 vs 23.5 ± 0.39; P < .05). NPs loaded with 17R-RvD1 resulted in a trend toward blunted vascular smooth muscle cell migration in a scratch assay. In a rat model of carotid angioplasty, 16-fold more NPs were present after treatment with TF-targeted NPs compared with scrambled NPs (P < .01), with a corresponding trend toward higher tissue levels of 17R-RvD1 (P = .06). Benzo-RvD1 was also detectable in arteries treated with targeted NP delivery and accumulated at 10 times higher levels than NP loaded with 17R-RvD1. There was a trend toward decreased CD45 immunostaining in vessels treated with NP containing benzo-RvD1 (0.76 ± 0.38 cells/mm2 vs 122.1 ± 22.26 cells/mm2; P = .06). There were no significant differences in early arterial inflammatory and cytokine gene expression by reverse transcription-polymerase chain reaction. Conclusions: TF-targeting peptides enhanced NP-mediated delivery of SPM to injured artery. TF-targeted delivery of SPMs may be a promising therapeutic approach to attenuate the vascular injury response.

Injectable Devices for Delivery of Liquid or Solid Protein Formulations.

Sustained delivery of protein therapeutics remains a largely unsolved problem across anatomic locations. Miniaturized devices that can provide sustained delivery of protein formulations have the potential to address this challenge via minimally invasive administration. In particular, methodologies that can optimize protein formulation independent of device manufacture have the greatest potential to provide a platform suitable for wide applications. The techniques developed here demonstrate the fabrication of tubular devices for sustained release of protein therapeutics. Utilizing a dip-casting process, fine-scale tubes can be reliably produced with wall thickness down to 30 µm. Techniques were developed that enabled effective loading of either solid or liquid formulations, while maintaining a cylindrical form-factor compatible with placement in a 22-gauge needle. Further, highly compacted protein pellets that approach the expected density of the raw materials were produced with a diameter (∼300 µm) suitable for miniaturized devices. Release from a solid-loaded device was capable of sustaining release of a model protein in excess of 400 days. Given significant interest in ocular applications, intravitreal injection was demonstrated in a rabbit model with these devices. In addition, to simulate repeated injections in ocular applications, serial intravitreal injection of two devices in a rabbit model demonstrated acceptable ocular safety without significant intraocular inflammation from clinical exam and histology.

Local decorin delivery via hyaluronic acid microrods improves cardiac performance, ventricular remodeling after myocardial infarction.

Heart failure (HF) remains a global public health burden and often results following myocardial infarction (MI). Following injury, cardiac fibrosis forms in the myocardium which greatly hinders cellular function, survival, and recruitment, thus severely limits tissue regeneration. Here, we leverage biophysical microstructural cues made of hyaluronic acid (HA) loaded with the anti-fibrotic proteoglycan decorin to more robustly attenuate cardiac fibrosis after acute myocardial injury. Microrods showed decorin incorporation throughout the entirety of the hydrogel structures and exhibited first-order release kinetics in vitro. Intramyocardial injections of saline (n = 5), microrods (n = 7), decorin microrods (n = 10), and free decorin (n = 4) were performed in male rat models of ischemia-reperfusion MI to evaluate therapeutic effects on cardiac remodeling and function. Echocardiographic analysis demonstrated that rats treated with decorin microrods (5.21% ± 4.29%) exhibited significantly increased change in ejection fraction (EF) at 8 weeks post-MI compared to rats treated with saline (-4.18% ± 2.78%, p < 0.001) and free decorin (-3.42% ± 1.86%, p < 0.01). Trends in reduced end diastolic volume were also identified in decorin microrod-treated groups compared to those treated with saline, microrods, and free decorin, indicating favorable ventricular remodeling. Quantitative analysis of histology and immunofluorescence staining showed that treatment with decorin microrods reduced cardiac fibrosis (p < 0.05) and cardiomyocyte hypertrophy (p < 0.05) at 8 weeks post-MI compared to saline control. Together, this work aims to contribute important knowledge to guide rationally designed biomaterial development that may be used to successfully treat cardiovascular diseases.

Precise surface functionalization of PLGA particles for human T cell modulation.

The biofunctionalization of synthetic materials has extensive utility for biomedical applications, but approaches to bioconjugation typically show insufficient efficiency and controllability. We recently developed an approach by building synthetic DNA scaffolds on biomaterial surfaces that enables the precise control of cargo density and ratio, thus improving the assembly and organization of functional cargos. We used this approach to show that the modulation and phenotypic adaptation of immune cells can be regulated using our precisely functionalized biomaterials. Here, we describe the three key procedures, including the fabrication of polymeric particles engrafted with short DNA scaffolds, the attachment of functional cargos with complementary DNA strands, and the surface assembly control and quantification. We also explain the critical checkpoints needed to ensure the overall quality and expected characteristics of the biological product. We provide additional experimental design considerations for modifying the approach by varying the material composition, size or cargo types. As an example, we cover the use of the protocol for human primary T cell activation and for the identification of parameters that affect ex vivo T cell manufacturing. The protocol requires users with diverse expertise ranging from synthetic materials to bioconjugation chemistry to immunology. The fabrication procedures and validation assays to design high-fidelity DNA-scaffolded biomaterials typically require 8 d.

Utility of transesophageal echocardiography during orthotopic liver transplantation: A narrative review.

Orthotopic liver transplantation (OLT) is the standard of care for patients suffering from end stage liver disease (ESLD). This is a high-risk procedure with the potential for hemorrhage, large shifts in preload and afterload, and release of vasoactive mediators that can have profound effects on hemodynamic equilibrium. In addition, patients with ESLD can have preexisting coronary artery disease, cirrhotic cardiomyopathy, porto-pulomary hypertension and imbalanced coagulation. As cardiovascular involvement is invariable and patient are at an appreciable risk of intraoperative cardiac arrest, Trans esophageal echocardiography (TEE) is increasingly becoming a routinely utilized monitor during OLT in patients without contraindications to its use. A comprehensive TEE assessment performed by trained operators provides a wealth of information on baseline cardiac function, while a focused study specific for the ESLD patients can help in prompt diagnosis and treatment of critical events. Future studies utilizing TEE will eventually optimize examination safety, quality, permit patient risk stratification, provide intraoperative guidance, and allow for evaluation of graft vasculature.

Robotic self-modulation enhances implantable long-acting drug delivery devices.

Integrating fibrotic capsule sensing with soft robotics may boost long-term performance of implantable drug delivery devices.

Replenishable prevascularized cell encapsulation devices increase graft survival and function in the subcutaneous space.

Beta cell replacement therapy (BCRT) for patients with type 1 diabetes (T1D) improves blood glucose regulation by replenishing the endogenous beta cells destroyed by autoimmune attack. Several limitations, including immune isolation, prevent this therapy from reaching its full potential. Cell encapsulation devices used for BCRT provide a protective physical barrier for insulin-producing beta cells, thereby protecting transplanted cells from immune attack. However, poor device engraftment posttransplantation leads to nutrient deprivation and hypoxia, causing metabolic strain on transplanted beta cells. Prevascularization of encapsulation devices at the transplantation site can help establish a host vascular network around the implant, increasing solute transport to the encapsulated cells. Here, we present a replenishable prevascularized implantation methodology (RPVIM) that allows for the vascular integration of replenishable encapsulation devices in the subcutaneous space. Empty encapsulation devices were vascularized for 14 days, after which insulin-producing cells were inserted without disrupting the surrounding vasculature. The RPVIM devices were compared with nonprevascularized devices (Standard Implantation Methodology [SIM]) and previously established prevascularized devices (Standard Prevascularization Implantation Methodology [SPVIM]). Results show that over 75% of RPVIM devices containing stem cell-derived insulin-producing beta cell clusters showed a signal after 28 days of implantation in subcutaneous space. Notably, not only was the percent of RPVIM devices showing signal significantly greater than SIM and SPVIM devices, but the intraperitoneal glucose tolerance tests and histological analyses showed that encapsulated stem-cell derived insulin-producing beta cell clusters retained their function in the RPVIM devices, which is crucial for the successful management of T1D.

Harnessing Biomaterials for Immunomodulatory-Driven Tissue Engineering.

Abstract: The immune system plays a crucial role during tissue repair and wound healing processes. Biomaterials have been leveraged to assist in this in situ tissue regeneration process to dampen the foreign body response by evading or suppressing the immune system. An emerging paradigm within regenerative medicine is to use biomaterials to influence the immune system and create a pro-reparative microenvironment to instigate endogenously driven tissue repair. In this review, we discuss recent studies that focus on immunomodulation of innate and adaptive immune cells for tissue engineering applications through four biomaterial-based mechanisms of action: biophysical cues, chemical modifications, drug delivery, and sequestration. These materials enable augmented regeneration in various contexts, including vascularization, bone repair, wound healing, and autoimmune regulation. While further understanding of immune-material interactions is needed to design the next generation of immunomodulatory biomaterials, these materials have already demonstrated great promise for regenerative medicine. Lay Summary: The immune system plays an important role in tissue repair. Many biomaterial strategies have been used to promote tissue repair, and recent work in this area has looked into the possibility of doing repair by tuning. Thus, we examined the literature for recent works showcasing the efficacy of these approaches in animal models of injuries. In these studies, we found that biomaterials successfully tuned the immune response and improved the repair of various tissues. This highlights the promise of immune-modulating material strategies to improve tissue repair.

Encapsulation of ß-NGF in injectable microrods for localized delivery accelerates endochondral fracture repair.

Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.

Local delivery of decorin via hyaluronic acid microrods improves cardiac performance and ventricular remodeling after myocardial infarction.

Heart failure (HF) is a global public health burden and associated with significant morbidity and mortality. HF can result as a complication following myocardial infarction (MI), with cardiac fibrosis forming in the myocardium as a response to injury. The dense, avascular scar tissue that develops in the myocardium after injury following MI creates an inhospitable microenvironment that hinders cellular function, survival, and recruitment, thus severely limiting tissue regeneration. We have previously demonstrated the ability of hyaluronic acid (HA) polymer microrods to modulate fibroblast phenotype using discrete biophysical cues and to improve cardiac outcomes after implantation in rodent models of ischemia-reperfusion MI injury. Here, we developed a dual-pronged biochemical and biophysical therapeutic strategy leveraging bioactive microrods to more robustly attenuate cardiac fibrosis after acute myocardial injury. Incorporation of the anti-fibrotic proteoglycan decorin within microrods led to sustained release of decorin over one month in vitro and after implantation, resulted in marked improvement in cardiac function and ventricular remodeling, along with decreased fibrosis and cardiomyocyte hypertrophy. Together, this body of work aims to contribute important knowledge to help develop rationally designed engineered biomaterials that may be used to successfully treat cardiovascular diseases.

Calcium Phosphate Delivery Systems for Regeneration and Biomineralization of Mineralized Tissues of the Craniofacial Complex.

Calcium phosphate (CaP)-based materials have been extensively used for mineralized tissues in the craniofacial complex. Owing to their excellent biocompatibility, biodegradability, and inherent osteoconductive nature, their use as delivery systems for drugs and bioactive factors has several advantages. Of the three mineralized tissues in the craniofacial complex (bone, dentin, and enamel), only bone and dentin have some regenerative properties that can diminish due to disease and severe injuries. Therefore, targeting these regenerative tissues with CaP delivery systems carrying relevant drugs, morphogenic factors, and ions is imperative to improve tissue health in the mineralized tissue engineering field. In this review, the use of CaP-based microparticles, nanoparticles, and polymer-induced liquid precursor (PILPs) amorphous CaP nanodroplets for delivery to craniofacial bone and dentin are discussed. The use of these various form factors to obtain either a high local concentration of cargo at the macroscale and/or to deliver cargos precisely to nanoscale structures is also described. Finally, perspectives on the field using these CaP materials and next steps for the future delivery to the craniofacial complex are presented.

Codelivery of synergistic antimicrobials with polyelectrolyte nanocomplexes to treat bacterial biofilms and lung infections.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

Impact of Microdevice Geometry on Transit and Retention in the Murine Gastrointestinal Tract.

Oral protein delivery technologies often depend on encapsulating or enclosing the protein cargo to protect it against pH-driven degradation in the stomach or enzymatic digestion in the small intestine. An emergent methodology is to encapsulate therapeutics in microscale, asymmetric, planar microparticles, referred to as microdevices. Previous work has shown that, compared to spherical particles, planar microdevices have longer residence times in the GI tract, but it remains unclear how specific design choices (e.g., material selection, particle diameter) impact microdevice behavior in vivo. Recent advances in microdevice fabrication through picoliter printing have expanded the range of device sizes that can be fabricated in a rapid manner. However, relatively little work has explored how device size governs their behavior in the intestinal environment. In this study, we probe the impact of geometry of planar microdevices on their transit and accumulation in the murine GI tract. Additionally, we present a strategy to label, image, and quantify these distributions in intact tissue in a continuous manner, enabling a more detailed understanding of device distribution and transit kinetics than previously possible. We show that smaller particles (194.6 ± 7 µm.diameter) tend to empty from the stomach faster than midsize (293.2 ± 7 µm.diameter) and larger devices (440.9 ± 9 µm.diameter) and that larger devices distribute more broadly in the GI tract and exit slower than other geometries. In general, we observed an inverse correlation between device diameter and GI transit rate. These results inform the future design of drug delivery systems, using particle geometry as an engineering design parameter to control device accumulation and distribution in the GI tract. Additionally, our image analysis process provides greater insight into the tissue level distribution and transit of particle populations. Using this technique, we demonstrate that microdevices act and translocate independently, as opposed to transiting in one homogeneous mass, meaning that target sites will likely be exposed to devices multiple times over the course of hours post administration. This imaging technique and associated findings enable data-informed design of future particle delivery systems, allowing orthogonal control of transit and distribution kinetics in vivo independent of material and cargo selection.